Albright hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterized by short stature, obesity, subcutaneous ossifications and brachydactyly. Some family members have these features in association with resistance to multiple hormones which activate Gs-coupled receptors (pseudohypoparathyroidism type Ia, PHP Ia) while others present with the somatic features alone (pseudopseudohypoparathyroidism, PPHP). Peripheral tissues from most affected patients have a 50% deficiency in Gs-alpha subunit function and/or expression in peripheral tissues (both PHP Ia and PPHP). We have identified a number of heterozygous inactivating mutations of the Gs-alpha gene in affected patients (both PHP Ia and PPHP). It has been proposed that tissue-specific imprinting of the Gs-alpha gene may explain the observation that maternal transmission of the Gs defect leads to offspring with PHP Ia while paternal transmission leads to PPHP. We have confirmed such a model in mice with a genetic knockout of the Gs-alpha gene (Gnas). Heterozygotes that inherit the knockout paternally are leaner than normal and are hypermetabolic and hyperactive, while mice which inherit the knockout maternally become obese and are hypometabolic and hypoactive. Both groups of mice have increased sensitivity to insulin based upon fasting glucose and insulin levels and the results of insulin and glucose tolerance tests. This is associated with increased insulin-stimulated glucose uptake in isolated skeletal muscle. This change does not appear to be caused by increased expression of the insulin-sensitive glucose transporter GLUT4. We are presently examining the activation of the insulin signaling pathway in liver and muscle from these mice, and are developing more tissue-specific knockouts of Gs-alpha. We and others have shown that in fact the Gs-alpha gene (Gnas) produces several products due to the use of alternative promoters and first exons, some which are maternally imprinted and others which are paternally imprinted. We have shown that the Gs-alpha promoter is not methylated but a region just upstream of the promoter is methylated only on the maternal allele. Within this differentially methylated region (DMR) is an alternative promoter and first exon (exon 1A) which is transcriptionally active only on the paternal allele. We showed that in patients with pseudohypoparathyroidism type Ib (PHP Ib), which is characterized by renal resistance to parathyroid hormone in the absence of other features, the exon 1A DMR has a paternal-specific imprinting pattern (unmethylated, transcriptionally active) on both alleles, indicating that this region is important for the pathogenesis of PHP Ib and the tissue-specific imprinting of Gs-alpha. We also showed that some PHPIb patients are lacking the gene product NESP55 due to methylation of the NESP55 promoter on both alleles. We are presently searching for mutations which might lead to the imprinting defect and are generating mouse models to study the role of the exon 1A DMR in Gnas imprinting.